This project has three goals. The first is to elucidate in detail the mechanism by which the bacterial enhancer-binding protein NTRC activates transcription initiation by the alternative holoenzyme form of RNA polymerase, sigma54-holoenzyme. The second is to study the sensing circuit that controls NTRC function in response to availability of combined nitrogen, and the third is to elucidate the physiological roles of the two central intermediates of nitrogen metabolism, glutamate and glutamine, and their precursor 2-oxoglutarate. The proposed studies will contribute to an understanding of transcriptional enhancers and enhancer-binding proteins generally, these being critical to the normal metabolism and development of humans and other eukaryotes . Further, they will contribute to understanding of nitrogen regulation, osmoregulation, and mechanisms for integrating these two major metabolic regulatory circuits. NTRC contacts sigma54-holoenzyme from its distant enhancer sites by DNA loop formation and must hydrolyze ATP to allow the polymerase to isomerize from closed to open complexes at a promoter. Specific aims with respect to the first goal are: a) to develop assays for the protein-protein contacts between NTRC and polymerase that mediate open complex formation and to define these contacts; b) to determine whether the requirement for ATP hydrolysis is thermodynamic as well as kinetic -- that is, whether the position of equilibrium favors closed complexes; c) to determine whether the mechanism for energy coupling depends on a transition between conformations of NTRC or on transient incorporation of phosphate into polymerase, as assessed by monitoring the configuration around the gamma phosphorus of ATP; d) to determine what step in the overall isomerization to open complexes is controlled by ATP hydrolysis, in particular, whether it is the isomerization of initial closed complexes to "second or intermediate" closed complexes. Specific aims with respect to the second goal are: a) to develop a reliable method for measuring 2-oxoglutarate pools, as we have done for measuring pools of glutamate and glutamine; b) to determine whether the in vivo rate of glnA transcription is controlled by a ratio of glutamine/2-oxoglutarate and, if so, how much this ratio varies under the full range of transcription rates. Specific aims with respect to the third goal are: a) to test the hypothesis that enteric bacteria perceive external nitrogen limitation as a limitation in the internal pool size of glutamine; b) to determine the role of the glutamate pool in osmoregulation. Srains with low glutamate pools grow slowly at intermediate and high external osmolarities. Is their internal pH abnormally alkaline under these conditions? Are their K+ pools low? Are their pools of trehalose elevated? Can the osmoprotectants proline and glycine-betaine substitute for (K+) glutamate?